Pluripotent stem cells have generated tremendous interest in the biomedical community. With the realization that stem cells can be isolated from many adult tissues has come the hope that cultures of relatively pure stem cells can be maintained in vitro for use in treating a wide range of conditions. Stem cells, with their capability for self-regeneration in vitro and their ability to produce differentiated cell types, may be useful for replacing the function of aging or failing cells in nearly any organ system. By some estimates, over 100 million Americans suffer from disorders that might be alleviated by tranplantation technologies that utilize stem cells (Perry (2000) Science 287:1423). Such illnesses include, for example, cardiovascular diseases, autoimmune diseases, diabetes, osteoporosis, cancers and burns.
Insulin-dependent diabetes mellitus (IDDM) is a good example of a disease that could be cured or ameliorated through the use of stem cells. Insulin-dependent diabetes mellitus is a disease characterized by elevated blood glucose and the absence of the hormone insulin. The cause of the raised sugar levels is insufficient secretion of the hormone insulin by the pancreas. In the absence of this hormone, the body""s cells are not able to absorb sugar from the blood stream in normal fashion, and the excess sugar accumulates in the blood. Chronically elevated blood glucose damages tissues and organs. IDDM is treated with insulin injections. The size and timing of insulin injections are influenced by measurements of blood sugar.
There are over 400 million diabetics in the world today. Diabetes is one of the most prevalent chronic diseases in the United States, and a leading cause of death. Estimates based on the 1993 National Health Interview Survey (NHIS) indicate that diabetes has been diagnosed in 1% of the U.S. population age  less than 45 years, 6.2% of those age 45-64 years, and 10.4% of those age  greater than 65 years. In other terms, in 1993 an estimated 7.8 million persons in the United States were reported to have this chronic condition. In addition, based on the annual incidence rates for diabetes, it is estimated that about 625,000 new cases of diabetes are diagnosed each year, including 595,000 cases of non-insulin-dependent diabetes mellitus (NIDDM) and 30,000 cases of insulin-dependent diabetes mellitus (IDDM). Persons with diabetes are at risk for major complications, including diabetic ketoacidosis, end-stage renal disease, diabetic retinopathy and amputation. There are also a host of less directly related conditions, such as hypertension, heart disease, peripheral vascular disease and infections, for which persons with diabetes are at substantially increased risk.
While medications such as injectable insulin and oral hypoglycemics allow diabetics to live longer, diabetes remains the third major killer, after heart disease and cancer. Diabetes is also a very disabling disease, because medications do not control blood sugar levels well enough to prevent swinging between high and low blood sugar levels, with resulting damage to the kidneys, eyes, and blood vessels.
Replenishment of functional glucose-sensing, insulin-secreting pancreatic beta cells through islet transplantation has been a longstanding therapeutic target. The limiting factor in this approach is the availability of an islet source that is safe, reproducible, and abundant. Current methodologies use either cadaverous material or porcine islets as transplant substrates (Korbutt et al., 1997). However, significant problems to overcome are the low availability of donor tissue, the variability and low yield of islets obtained via dissociation, and the enzymatic and physical damage that may occur as a result of the isolation process (reviewed by Secchi et al., 1997; Sutherland et al., 1998). In addition are issues of immune rejection and current concerns with xenotransplantation using porcine islets (reviewed by Weir and Bonner-Weir, 1997).
As a further example, stem cells capable of generating blood cells would also be of tremendous value for treatment of several diseases. A number of diseases or conditions result frown inappropriate levels or inadequate function of blood platelets. For example, xe2x80x9cthrombocytopeniasxe2x80x9d are the result of an abnormally small number of platelets in the circulating blood. Thombocytopenia can be due to antibody mediated platelet destruction, massive blood transfusions, cardio-pulmonary by-pass or bone marrow failure from malignant infiltration, aplastic anemia or chemotherapy. xe2x80x9cThrombocythemicxe2x80x9d disorders, on the other hand, are the result of a high platelet count. Finally, xe2x80x9cthrombocytopathicxe2x80x9d blood disorders are characterized by abnormally low or high platelet function, although platelet counts may be normal. Blood platelets are required for the maintenance of normal hemostasis. Platelets initiate blood clot formation and release growth factors that speed the process of wound healing as well as potentially serving other functions. Blood platelets are circulating cells that are crucial for the prevention of bleeding and for blood coagulation. Megakaryocytes are the cellular source of platelets and arise from a common bone marrow precursor cell which gives rise to all hematopoietic cell lineages. Stem cells could be used to generate cells in vitro or could be implanted to provide a stable source of cells capable of producing platelets.
In addition, extensive radiation therapy is used to treat many cancers. The radiation is lethal to the patient""s endogenous bone marrow stem cells. Currently, these are replaced by transplantation in a procedure fraught with complications. An abundant supply of hematopoietic stem cells could be used for repeated treatments to replenish the depleted endogenous cells.
Many neural disorders are marked by death of nerve cells. Adult nerve cells regenerate poorly and nerve death often causes irreparable damage to congnitive and sensorimotor functions. There has been some success in treating disorders caused by nerve death with transplants of fetal nerve tissue. Fetal tissue has a greater ability to take up residence in the adult brain and differentiate into the appropriate cell type. However, obtaining sufficient fetal tissue is difficult and presents many ethical problems. Neural stem cells are capable of differentiating into many cell types of the nervous system. Remarkably, some neural stem cells are capable of migrating through the brain and settling in regions of nerve cell death. Such cells may then generate new neural processes to integrate with the endogenous neural network. It is expected that neural stem cells can be used to treat disorders such as Alzheimer""s disease, Parkinson""s disease, stroke, ischemia, trauma, spinal cord injuries, damage from infectious disease etc.
It is an object of the present invention to provide simple methods for the isolation and propagation of stem cells from virtually any tissue type. Such stem cells can then be used, for example, for direct transplantation or to produce differentiated cells in vitro for transplantation or. The invention accordingly provides, for example, pancreatic and hepatic stem cells that may serve as a source for many other, more differentiated cell types such as pancreatic beta cells. Advantages lie in obviating the need for physical dissociation of tissue in order to obtain differentiated cells for various uses, and the potential for greater reproducibility and control of the process. With respect to pancreatic cells, successful achievement requires the differentiation and maturation of glucose-sensing, insulin-secreting beta cells from an expandable precursor population.
The present invention relates to materials and methods for obtaining substantially pure populations of animal stem or progenitor cells. The invention further provides animal stem or progenitor cell populations and cell derivatives thereof, as well as method of using these cell populations.
In a preferred embodiment the invention provides a method for preparing a substantially pure non-adherent population of progenitor cells which is at least about 50%, but more preferably about 60%, 70%, 80% or most preferably about 90% pure. In certain embodiments, the population of progenitor cells is obtained from an animal tissue, preferably a mammalian organ or other mammalian tissue, which is disrupted by mechanical or enzymatic means so as to yield a cell population which includes at least one progenitor cell. In preferred embodiments, the tissue is human tissue. The animal tissue may be any adult or embryonic tissue including, but not exclusive to: pancreatic tissue, liver tissue, smooth muscle tissue, striated muscle tissue, cardiac muscle tissue, bone tissue, bone marrow tissue, bone spongy tissue, cartilage tissue, liver tissue, pancreas tissue, pancreatic ductal tissue, spleen tissue, thymus tissue, tonsil tissue, Peyer""s patch tissue, lymph nodes tissue, thyroid tissue, epidermis tissue, dermis tissue, subcutaneous tissue, heart tissue, lung tissue, vascular tissue, endothelial tissue, blood cells, bladder tissue, kidney tissue, digestive tract tissue, esophagus tissue, stomach tissue, small intestine tissue, large intestine tissue, adipose tissue, uterus tissue, lung tissue, testicular tissue, ovarian tissue, prostate tissue, connective tissue, endocrine tissue, mesentery tissue, fetal tissue and umbilical tissue. In certain embodiments, the tissue is a non-neuronal animal tissue which does not include brain or central nervous system tissue. Preferably the enrichment of stem/progenitor cells from the original cell suspension obtained from the tissue is at least about 100-fold, but more preferably is at least about 1000-fold.
In certain preferred embodiments, the cell suspension derived from this animal tissue is then treated with a growth factor preparation which may include any of a number of different growth factors including epidermal growth factor, transforming growth factor, hepatocyte growth factor, fibroblast growth factor, leukemia inhibitory factor, insulin-like growth factor and platelet-derived growth factor.
The progenitor cell population within the animal cell suspension is then allowed to proliferate in the presence of the growth factor population and takes on a non-adherent, floating characteristic. In certain instances, the progenitor cell population form homotypic cell spheres. The phenotypic characteristics of the progenitor cell population provide both an indication that the cell suspension has become enriched in the stem/progenitor cell population as well as providing certain physical features which may be used to enrich for the stem/progenitor cells.
The invention further provides certain markers, including c-kit, Sca and Nestin, for identifying and/or enriching the population of stem/progenitor cells. The invention still further provides for derivatives of these stem/progenitor populations which can be obtained under proper conditions. The stem/progenitor cell derivatives may express a marker such as Pdx-1, glucagon, or insulin.
The present invention further relates to substantially pure preparations of viable pancreatic progenitor cells, and methods for isolating such cells from essentially any tissue, notably liver, muscle and pancreatic tissue. The present invention further concerns certain uses for such progenitor cells, and their progeny.
In general, the invention features a cellular composition including, as the cellular component, a substantially pure population of viable pancreatic progenitor cells which progenitor cells are capable of proliferation in a culture medium. In a preferred embodiment, the cellular composition has fewer than about 20%, more preferably fewer than about 10%, most preferably fewer than about 5% of lineage committed cells.
In one embodiment, the progenitor cells of the present invention are characterized by an ability for self-regeneration in a culture medium and differentiation to pancreatic lineages. In a preferred embodiment, the progenitor cells are inducible to differentiate into pancreatic islet cells, e.g., xcex2 islet cells, xcex1 islet cells, xcex4 islet cells, or xcfx86 islet cells. Such pancreatic progenitor cells may be characterized in certain circumstances by the expression of one or more of: homeodomain type transcription factors such as STF-1; PAX gene(s) such as PAX6; PTF-1; hXBP-1; HNF genes(s); villin; tyrosine hydroxylase; insulin; glucagon; and/or neuropeptide Y. The pancreatic progenitor cells of the present invention may also be characterized by binding to lectin(s), and preferably to a plant lectin, and more preferably to peanut agglutinin. In certain preferred embodiments, the progenitor cells are PDX1+, e.g., by FACS sorting, and capable of differentiation into glucose-responsive insulin secreting cells. In certain preferred embodiments, the progenitor cells are PDX1+ and Glut2+. In certain preferred embodiments, the progenitor cells are PDX1+, Glut2+ and stain with PNA.
The invention provides multiple methods for obtaining pancreatic progenitor cells. In one embodiment, the cells are obtained by propagation in a non-adherent culture. In another embodiment, the cells are obtained as NACs arising from an adherent culture.
In certain preferred embodiments, the subject pancreatic progenitor cells will have one or more of the following characteristics: (i) able to grow in 2-5 percent fetal calf serum; (ii) able to grow on plastic, e.g., no need to use matrigel; (iii) no statistically significant induction of cells to proliferate or differentiate when treated with TGFxcex25 (GenBank accession P16176) at concenrates up to 30 pg/ml.
In yet another embodiment, the invention features a pharmaceutical composition including as the cellular component, a substantially pure population of viable pancreatic progenitor cells, which progenitor cells are capable of proliferation in a culture medium.
In general, the preferred progenitor cells will be of mammalian origin, e.g., cells isolated from a primate such as a human, from a miniature swine, or from a transgenic mammal, or are the cell culture progeny of such cells. In one embodiment, pancreatic ductual tissue is isolated from a patient and subjected to the present method in order to provide a resulting culture of pancreatic progenitor cells (or differentiated cells derived therefrom). Gene replacement or other gene therapy is carried out ex vivo, and the isolated cells are transplanted back into the initial donor patient or into a second host patient.
In another aspect, the invention features a cellular composition comprising, as a cellular population, at least 50% (though more preferably at least 75, 90 or 95%) progenitor cells and capable of self-regeneration in a culture medium.
In yet another aspect, the invention features a cellular composition consisting essentially of, as the cellular population, viable pancreatic progenitor cells capable of self-regeneration in a culture medium and differentiation to pancreatic lineages. For instance, in certain embodiments the progenitor cells are isolated from pancreatic intralobular duct explants, e.g. isolated by biopsy, or are the cell culture progeny of such cells.
Several aspects of the invention feature a method for isolating pancreatic progenitor cells from a sample of pancreatic duct. In general, the method provides for a culture system that allows reproducible expansion of pancreatic ductual epithelium while maintaining xe2x80x9cstemmednessxe2x80x9d and the ability to differentiate into endocrine and exocrine cells. In one embodiment, pancreatic ductal tissue is treated with digestive enzymes to produce a cell suspension. The cell suspension is cultured in a non-adherent culture vessel in the presence of various growth factors. In certain embodimetns, propagating cells give rise to spheres of cells that are capable of differentiating into a wide range of cell types. In another instance, pancreatic ductal epithelium is obtained, e.g., by explant or enzymatic digestion, and cultured to confluence. The confluent cell population is contacted with an agent, e.g., a trophic agent such as a growth factor, which causes differentiation of progenitor cells in the cultured population. Subsequently, progenitor cells from the explant that proliferate in response to the agent are isolated, such as by direct mechanical separation of newly emerging buds from the rest of the explant or by dissolution of all or a portion of the explant and subsequent isolation of the progenitor cell population.
In certain embodiments, the culture is contacted with a cAMP elevating agents, such as 8-(4-chlorophenylthio)-adenosine-3xe2x80x2:5xe2x80x2-cyclic-monophosphate (CPT-cAMP) (see, for example, Koike Prog. Neuro-Psychopharmacol. and Biol. Psychiat. 16 95-106 (1992)), CPT-cAMP, forskolin, Na-Butyrate, isobutyl methylxanthine (IBMX) and cholera toxin (see Martin et al. J. Neurobiol. 23 1205-1220 (1992)) and 8-bromo-cAMP, dibutyryl-cAMP and dioctanoyl-cAMP (e.g., see Rydel et al. PNAS 85:1257 (1988)).
In certain embodiments, the culture is contacted with a growth factor, e.g., a mitogenic growth factor, e.g., the growth factor is selected from a group consisting of IGF, TGF, FGF, EGF, HGF, hedgehog or VEGF. In other embodiments, the growth factor is a member of the TGFxcex2 superfamily, preferably of the DVR (dpp and vg1 related) family, e.g., BMP2 and/or BMP7.
In certain embodiments, the culture is contacted with a steroid or corticosteroid such as, for example, hydrocortisone, deoxyhydrocortisone, fludrocortisone, prednisolone, methylprednisolone, prednisone, triamcinolone, dexamethasone, betamethasone and paramethasone. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., pp. 1239-1267 and 2497-2506, Berkow et al., eds., Rahway, N.J., 1987).
In a preferred embodiment, the cultures are contacted with a cAMP elevating agent, a growth factor and a steroid or corticosteroid, e.g., with the DCE cocktail described herein.
In another aspect, the invention features, a method for screening a compound for ability to modulate one of growth, proliferation, and/or differentiation of progenitor cells obtained by the subject method, including: (i) establishing an isolated population of pancreatic progenitor cells; (ii) contacting the population of cells with a test compound; and (iii) detecting one of growth, proliferation, and/or differentiation of the progenitor cells in the population, wherein a statistically significant change in the extent of one of growth, proliferation, and/or differentiation in the presence of the test compound relative to the extent of one of growth, proliferation, and/or differentiation in the absence of the test compound indicates the ability of the test compound to modulate one of the growth, proliferation, and/or differentiation.
In another aspect, the invention features, a method for treating a disorder characterized by insufficient insulin activity, in a subject, including introducing into the subject a pharmaceutical composition including pancreatic progenitor cells derived by the subject method, or differentiated cells arising therefrom, and a pharmaceutically acceptable carrier. In preferred embodiments, the progenitor cells are derived from a donor source (which may also be the transplant patient), and expanded at least order of magnitude prior to implantation. As shown in FIG. 40, the subject cellular compositions can be used to rescue diabetic mice.
In a preferred embodiment the subject is a mammal, e.g., a primate, e.g., a human.
In another preferred embodiment the disorder is an insulin dependent diabetes, e.g., type I diabetes.
In another aspect the invention provides differentiation media for promoting differentiation of progenitor cells into differentiated pancreatic cell types. In one embodiment, the invention comprises a pancreatic differentiation medium comprising a cAMP elevating agent, PYY and fetal bovine serum. In preferred embodiments the medium comprises forskolin. A particularly preferred pancreatic differentiation medium comprises at least 25 mM forskolin, at least 150 ng/ml PYY and at least 3% fetal bovine serum.
In a further aspect, the invention provides methods for obtaining differentiated pancreatic cell types. In one embodiment, the method for obtaining differentiated pancreatic cell types comprises obtaining a cell suspension from an animal tissue, treating the cell suspension with a growth factor preparation, allowing proliferation of non-adherent cells and contacting said proliferated non-adherent cells with a differentiation medium and an adherent matrix. Under such conditions, non-adherent cells adhere to the matrix and give rise to at least one differentiated pancreatic cell type. In preferred embodiments, progenitor cells are obtained from post-natal human tissue. In preferred embodiments, the growth factor preparation comprises one or more of the following: epidermal growth factor, basic fibroblast growth factor, hepatocyte growth factor, transforming growth factor alpha, insulin-like growth factor I and insulin-like growth factor II. The methods of the invention permit the formation of both exocrine and endocrine cell types. Exocrine cell types are preferably expresse carboxypeptidase A. Endocrine cell types may include glucagon-expressing cells, insulin-expressing cells and somatostatin expressing cells. Adherent matrices of the method are preferably matrices derived from a cancerous cell line and preferably a sarcoma or a bladder carcinoma. Notably, the methods of the invention permit the formation of insulin producing cells that are capable of producing insulin in a glucose-regulated manner.
In a further embodiment, the invention provides methods of obtaining glucagon-expressing cells, comprising propagating a progenitor cell in the presence of 15% KO-SR; and differentiating the cell by contacting it with an adherent matrix and a differentiation medium. Under such conditions, the progenitor cell gives rise to glucagon expressing cells.
In yet another embodiment, the invention provides a method of obtaining somatostatin-expressing cells, comprising propagating a progenitor cell in the presence of LIF and differentiating the cell by contacting it with an adherent matrix and a differentiation medium. Under these conditions, the progenitor cell gives rise to somatostatin expressing cells.
In yet another embodiment, the pancreatic progenitor cells are induced to differentiate into pancreatic islet cells, e.g., xcex2 islet cells, xcex1 islet cells, xcex4 islet cells, or xcfx86 islet cells, subsequent to being introduced into the subject.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.